Lignocellulosic biomass is widely recognized as a promising source of raw material for production of renewable fuels and chemicals. The primary obstacle impeding the more widespread production of energy from biomass feedstocks is the general absence of low-cost technology for overcoming the recalcitrance of these materials to conversion into useful fuels. Lignocellulosic biomass contains carbohydrate fractions (e.g., cellulose and hemicellulose) that can be converted into ethanol. In order to convert these fractions, the cellulose and hemicellulose must ultimately be converted or hydrolyzed into monosaccharides; it is the hydrolysis that has historically proven to be problematic.
Biologically mediated processes are promising for energy conversion, in particular, for the conversion of lignocellulosic biomass into fuels. Biomass processing schemes involving enzymatic or microbial hydrolysis commonly involve four biologically mediated transformations: (1) the production of saccharolytic enzymes (cellulases and hemicellulases); (2) the hydrolysis of carbohydrate components present in pretreated biomass to sugars; (3) the fermentation of hexose sugars (e.g., glucose, mannose and galactose); and (4) the fermentation of pentose sugars (e.g., xylose and arabinose). These four transformations occur in a single step in a process configuration called consolidated bioprocessing (CBP), which is distinguished from other less highly integrated configurations in that it does not involve a dedicated process step for cellulase and/or hemicellulase production. CBP offers the potential for lower cost and higher efficiency than processes featuring dedicated cellulase production. The benefits result in part from avoided capital costs, substrate and other raw materials, and utilities associated with cellulase production.
Bakers' yeast (Saccharomyces cerevisiae or S. cerevisiae) remains the preferred microorganism for the production of ethanol (Van Zyl et al., Adv. Biochem. Eng. Biotechnol. 108:205-235, 2007). Attributes in favor of this microbe are (i) high productivity at close to theoretical yields (0.51 gram of ethanol produced/gram glucose used), (ii) high osmo- and ethanol-tolerance, (iii) natural robustness in industrial processes, (iv) being generally regarded as safe (GRAS) due to its long association with wine and bread making and beer brewing. Furthermore, S. cerevisiae exhibits tolerance to inhibitors commonly found in hydrolyzates resulting from biomass pretreatment. The major shortcoming of S. cerevisiae is its inability to utilize complex polysaccharides such as cellulose, or its break-down products, such as cellobiose and cellodextrins. One strategy for developing CBP-enabling microorganisms such as S. cerevisiae is by engineering them to express a heterologous cellulase and/or a hemicellulase system.
Three major types of enzymatic activities are required for native cellulose degradation. One type is endoglucanases (1,4-β-D-glucan 4-glucanohydrolases; Enzyme Commission (EC) 3.2.1.4). Endoglucanases (Eg or EG) cut at random in the cellulose polysaccharide chain of amorphous cellulose, generating oligosaccharides of varying lengths and consequently new chain ends. Another type is exoglucanases. Exogluconases include cellodextrinases (1,4-β-D-glucan glucanohydrolases; EC 3.2.1.74) and cellobiohydrolases (1,4-β-D-glucan cellobiohydrolases; EC 3.2.1.91). Exoglucanases act in a processive manner on the reducing or non-reducing ends of cellulose polysaccharide chains, liberating either glucose (glucanohydrolases) or cellobiose (cellobiohydrolase) as major products. Exoglucanases can also act on microcrystalline cellulose, presumably peeling cellulose chains from the microcrystalline structure. Classically, exoglucanases such as the cellobiohydrolases (CBHs) possess tunnel-like active sites, which can only accept a substrate chain via its terminal regions. These exo-acting CBH enzymes act by threading the cellulose chain through the tunnel, where successive cellobiose units are removed in a sequential manner. Sequential hydrolysis of a cellulose chain is termed “processivity.”
Yet another type is beta-glucosidases (beta glucoside glucohydrolases, β-glucosidases or BGLs; EC 3.2.1.21). BGLs play an important role in the hydrolysis of materials containing cellulose or soluble oligomers of glucose. There have been reports of the role and importance of BGLs during hydrolysis (see, e.g., Viikari et al., Adv. Biochem. Eng. Biotechnol., 108:121-145, 2007; and Bhatia et al., Crit. Rev. Biotechnol., 22:375-407, 2002). These enzymes typically act on soluble oligomers of glucose which are linked via beta 1-4 type bonds, including dimers (cellobiose) where they usually have highest activity, as well as longer chain oligomers where they are typically less active. Examples of BGL domains have been described and include, for example, a glycosyl hydrolase family 3 n-terminal domain, a glycosyl hydrolase family 3 c-terminal domain, and a fibronectin type III like domain.
Structurally, cellulases generally consist of a catalytic domain joined to a cellulose-binding module (CBM) via a linker region that is rich in proline and/or hydroxy-amino acids. In type I exoglucanases, the CBM domain is found at the C-terminal extremity of these enzyme (this short domain forms a hairpin loop structure stabilized by 2 disulfide bridges). In type 2 CBHs, the CBM is found at the N-terminus. In some cases, however, cellulases do not contain a CBM, and only contain a catalytic domain. Examples of such CBM-lacking cellulases include CBHs from Humicola grisea, Phanerochaete chrysosporium and Aspergillus niger. Grassick et al., Eur. J. Biochem., 271:4495-4506, 2004.
With the aid of recombinant DNA technology, several of these heterologous cellulases from bacterial and fungal sources have been transferred to S. cerevisiae, enabling the degradation of cellulosic derivatives (Van Rensburg et al., Yeast, 14:67-76, 1998), or growth on cellobiose (Van Rooyen et al., J. Biotech., 120:284-295, 2005; and McBride et al., Enzyme Microb. Techol. 37:93-101, 2005).
Related work was described by Fujita et al., (Appl. Environ. Microbiol., 70:1207-1212, 2004) where cellulases immobilized on the yeast cell surface had significant limitations. First, Fujita et al. were unable to achieve fermentation of amorphous cellulose using yeast expressing only recombinant Bgl1 and EgII. A second limitation of the Fujita et al. approach was that cells had to be pre-grown to high cell density on standard carbon sources before the cells were useful for ethanol production using amorphous cellulose (e.g., Fujita et al. uses high biomass loadings of ˜15 g/L to accomplish ethanol production).
As noted above, ethanol producing yeast such as S. cerevisiae require addition of external cellulases when cultivated on cellulosic substrates, such as pre-treated wood, because this yeast does not produce endogenous cellulases. Expression of fungal cellulases such as Trichoderma reesei (T. reesei) Cbh1 and Cbh2 in yeast S. cerevisiae have been shown to be functional. Den Haan et al., Enzyme and Microbial Technology, 40:1291-1299, 2007. However, current levels of expression and specific activity of cellulases heterologously expressed in yeast are still not sufficient to enable growth and ethanol production by yeast on cellulosic substrates without externally added enzymes. While studies have shown that perhaps certain cellulases, such as T. reesei Cbh1, have some activity when heterologously expressed, there remains a significant need for improvement in the specific activity of heterologously expressed cellulases in order to attain the goal of achieving a CBP system capable of efficiently and cost-effectively converting cellulosic substrates to ethanol.
Currently, there is no reliable way to predict which cellulases will be efficiently expressed in heterologous organisms. For example, despite the fact that T. reesei Cbh1 and T. emersonii Cbh1 are both endogenously expressed at high levels, heterologous expression of these proteins in yeast yielded disparate results. Also, Talaromyces emersonii (T. emersonii) Cbh1 expression in yeast was significantly greater in yeast than T. reesei Cbh1 under similar conditions. See Int'l Pub. No. WO 2009/138877. Efficient expression may depend, for example, on chaperone proteins that differ in the heterologous organisms and in the cellulase's native organism. Furthermore, even cellulases which are expressed at high levels may not be particularly active in a heterologous organism. For example, a cellulase may be subject to different post-translational modifications in the heterologous host organism than in the native organism from which the cellulase is derived. Protein folding and secretion can also be a barrier to heterologous cellulase expression.
Therefore, in order to address the limitations of heterologous cellulase expression in CBP systems, the present invention provides the expression of several BGLs in host cells, such as the yeast S. cerevisiae. The expression level and secreted activity level of the BGLs was characterized. In addition, the BGLs were purified and their specific activity on hardwood derived pretreated solids (C6 solids) and hardwood derived hemicellulose liquor (C5 liquor) was determined. The corresponding BGL genes, or variants and combinations thereof, in such host cells were well expressed and resulted in improved specific activity of the expressed BGLs. Also, the combination of purified BGLs with one or more other cellulases, or host cells expressing the BGLs and one or more other cellulases, also resulted in improved specific activity of the expressed BGLs. Thus, such genes and expression systems are useful for efficient and cost-effective CBP systems.